In vivo and extract analyses by one-dimensional 31P NMR have been a key tool in investigating energy-related metabolism. Although many phosphorylated metabolites have been observed, many of them have yet to be identified. This reflects the difficulty in identifying them using 31P NMR alone. Two-dimensional 1H-31P correlation experiments have been shown to be useful for assigning phosphorylated metabolites. To obtain better sensitivity and structure information, 1H-detected 31P-1H heteronuclear total correlation spectroscopy (heteroTOCSY) was implemented and a complete chemical shift assignment for a number of phosphorylated standards was made. The time courses of 1D heteroTOCSY signal intensity versus spin-locking time were established for these standards to aid the optimization of the 2D experiment. This method was applied to crayfish extracts for the assignment of glucose 6-phosphate, α-glycerophosphate, ribose 5-phosphate, fructose 1,6-bisphosphate, phosphocholine, phosphoethanolamine, glucose 1-phosphate, glycerophosphoethanolamine, glycerophosphocholine, ATP, ADP, and AMP. An alkyl phosphate, a hexose 1-phosphate, and a UDP-hexose were also observed. These assignments allowed the identification of many changes in the 31P MR spectra of crayfish extracts elicited by treatment with the organophosphate pesticide chlorpyrifos. The assignment of an in vivo 31P spectrum of a live crayfish was also made based on the extract assignment. This approach should be a powerful tool for examining stress-associated changes in the metabolism of phosphorylated compounds.
|Number of pages
|Published - Oct 15 1998
Bibliographical noteFunding Information:
1 This work was supported, in part, by the Medical Research Council of the United Kingdom and the University of California Davis Center for Ecological Health Research (U.S. EPA No. R819658). Although the information in this document has been funded wholly or in part by the U.S. EPA, it may not necessarily reflect the views of the agency and no official endorsement should be inferred.
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology